Materials and methods relating to the transfer of nucleic acid into quiescent cells

ABSTRACT

Materials and methods for transferring nucleic acid encoding a polypeptide for treating a disease or disorder into populations of quiescent cells such as haematopoietic stem cells (HSCs), using retroviral packaging cell lines and retroviral particles expressing and display a growth factor such as stem cell factor (SCF) on the cell surface or as a fusion with a viral envelope protein. The present invention also relates to compositions comprising the retroviral packaging cell lines and retroviral particles, and their use in methods of medical treatment, in vivo and ex vivo.

This is application is the U.S. national stage application ofInternational application Serial No. PCT/GB96/02405, filed Sep. 30,1996, which claimed priority from Great Britain patent applicationSerial No. 9519776.0, filed Sep. 28, 1995, and claims the benefit of thefiling dates thereof under 35 U.S.C. §119.

FIELD OF THE INVENTION

The present invention relates to materials and methods for transferringnucleic acid encoding a polypeptide for treating a disease or disorderinto populations of quiescent cells such as haematopoietic stem cells(HSCs), using retroviral packaging cell lines and retroviral particlesexpressing and displaying a growth factor such as stem cell factor (SCF)on the cell surface or as a fusion with a viral envelope protein. Thepresent invention also relates compositions comprising the retroviralpackaging cell lines and retroviral particles, and their use in methodsof medical treatment, in vivo and ex vivo.

BACKGROUND TO THE INVENTION

The development of somatic gene therapy as a treatment for single geneinherited diseases and some acquired conditions, such as certain typesof cancer, represents one of the most important technical advances inmedicine. Blood related disorders such as the X-linkedimmunodeficiencies, or chronic granulomatous disease (CGD), are amongstthe most favourable candidates as model systems for the evolution ofthis technology. The general feasibility of gene therapy for disordersof this type has been amply demonstrated by the results obtained in thetreatment adenosine deaminase dependent severe combined immunodeficiency(ADA-SCID) using peripheral blood T-cells.

However, many problems stand in the way of the realisation of thepromise of these techniques. For example, in the experiments describedabove, the T-cells including the genes required by the patients are notimmortal, requiring the therapy to be repeated at regular intervals.Further, attempts to effect a permanent correction, for example by genetransfer into pluripotent haematopoietic stem cells (PHSC), have thusfar been unsuccessful.

There are a number reasons for this. Firstly, PHSC are very rare in thebone marrow cell population, and so although work has been done on bonemarrow cell culture, it is very difficult to draw conclusions from thiswork regarding PHSCs. Further, in humans there is a dearth of markers toidentify PHSC and, at present, the most reliable marker of immaturehuman bone marrow cells is the CD34 antigen, which marks about 1-2% oftotal marrow cells. However, probably only about 0.1% of these CD34+cells are true PHSC. In addition, there are no wholly reliable assaysfor human PHSC, unlike murine systems, where the rescue of lethallyirradiated individuals can be used to test for PHSC.

Recently, a method to enrich for PHSC has been described by Beradi et al(Science, 267, 104-108, (1995)) which exploits the quiescence of PHSCsas a basis for their functional isolation. In this method, bone marrowcells were incubated for 7 days in the presence of the cytokines stemcell factor (SCF) and IL-3, to stimulate division in all of theprogenitor cells, but not in true PHSC. The cytotoxic agent,5-fluorouracil (5-FU), was then added to these cultures, resulting inthe death of all dividing cells in the culture. However, quiescentcells, including PHSC which average only 1 in 10⁵ of the original cells,were spared in this process. Accordingly, the authors reported obtainingan enriched population of cells having the characteristics of true PHSC.

However, the authors of this paper were unable to find any combinationof cytokines that was able to stimulate these cells to divide, otherthan incubation in long term marrow culture (LTC), which also leads totheir differentiation.

Thus, although, this method produces highly enriched populations ofPHSC, it is their quiescence, the very property exploited for theirisolation by Beradi et al, that still represents the most significanthurdle limiting current gene therapy protocols. This is because mosthighly developed vector systems presently used for gene transduction arebased on murine retroviruses and these viruses (and the vectors derivedfrom them) are unable to stably integrate their genome into non-dividingcells, rendering PHSCs refractory to retroviral gene transfer.

Previously, we presented an abstract at the European Working Group forGene Therapy in November 1994 disclosing that a retroviral cell linecontaining a viral vector incorporating nucleic acid encoding GCD andexpressing stem cell factor on its surface was able to achieve improvedrates of transduction in a bone marrow cell culture. However, asmentioned above this cell culture contains a very low proportion ofPHSC, and this treatment would not be expected to stimulate the PHSC todivide or to allow the stable integration of the nucleic acid encodingGCD into the PHSC genome. An important fact underlying this expectationis that in Beradi et al, stem cell factor was one of the cytokines usedto stimulate selectively division in the most of the cells in marrowcell culture (but not the PHSC), allowing them to be killed to leave theenriched population of stem cells.

SUMMARY OF THE INVENTION

The present invention is based on the unexpected finding that it ispossible to get haematopoeitic stem cells to cycle transiently duringthe period of exposure to vectors incorporating nucleic acid encoding adesired protein or polypeptide by exposing them to bound growth factorssuch as stem cell factor. This observation means that contrary to priorexpectations, a population of quiescent cells such as PHSC can be usedas targets for vectors incorporating nucleic acid encoding a desiredprotein or polypeptide, provided that the cells are additionally exposedto a surface bound growth factor, e.g. stem cell factor expressed by aretroviral packaging cell line so that it is bound on the cell surfaceor expressed as a fusion with an envelope protein of retrovirus so thatthe growth factor is displayed on the surface of the retrovirus.

Without wishing to be bound by any particular theory, we believe thatthe exposure of the quiescent cells to the membrane or surface boundgrowth factor induces them to start dividing, so that the nucleic acid,e.g. packaged in retroviral particles produced by a retroviral packagingcell line, can infect the cells and become incorporated into theirgenomes which become accessible during cellular division when thenuclear membrane dissolves. This method has the advantage that it can beadapted for the treatment of a wide variety of disorders, byincorporating nucleic acid encoding an appropriate protein orpolypeptide into the vector. A further advantage of the method is thatby stimulating the quiescent cells to differentiate at the time of genetransfer, preferential amplification of the transduced cells relative tothe non-transduced cells can be achieved.

Accordingly, in a first aspect, the present invention provides aretroviral packaging cell line transformed with a viral vectorcomprising nucleic acid encoding a polypeptide for treating a disease ordisorder, the retroviral packaging cell line being capable of expressingnucleic acid encoding a growth factor so that the growth factor is (i)displayed on the cell surface or (ii) expressed as a fusion with a viralenvelope protein so that the growth factor is displayed on the surfaceof viral particles,

wherein the cell line packages the nucleic acid encoding the polypeptidein viral particles produced by the retroviral packaging cell line, thecell line being for use in a method of medical treatment of a disease ordisorder that responds to the polypeptide.

In this aspect, the retroviral packaging cell line includes nucleic acidencoding viral envelope protein so that the cell line can produce viralparticles and package the nucleic acid encoding the polypeptide fortreating the disease or disorder in them.

In this application, “quiescent” refers to cells that are unlikely toenter mitosis within the next 24 hours in the absence of appropriategrowth stimulus. Preferably, the population of quiescent cells areenriched in haematopoeitic stem cells, for instance by employing theisolation method of Beradi et al (supra) using bone marrow cells. Otherquiescent cell types suitable for use in the invention include restingT-lymphocytes, B-lymphocytes and monocytes, stem cells ofnon-haematopoietic tissues such as liver and muscle, epithelial stemcells in skin, gut, bladder and airways, vascular endothelial cells,quiescent neoplastic cells and germ cells such as sperm progenitors.

In a further aspect, the present invention provides retroviral particlesdisplaying surface bound growth factor as a fusion with an envelopeprotein, the particles being produced by the retroviral packaging cellline as set out above.

In one embodiment, the surface bound growth factor is provided byengineering the retroviral packaging cell line to express growth factoron its surface by transfecting the cell line with nucleic acid encodingthe growth factor.

In an alternative embodiment, a retroviral vector expressing surfacebound growth factor (e.g. SCF) could be prepared by constructing apackaging cell line engineered to produce a chimeric retroviral envelopeprotein fused to all or part of the growth factor. The growth factor canbe used to replace the natural binding domain of the envelope protein,or can be fused directly to the C- or N- terminus of a retroviralenvelope protein. Such chimeric envelopes have been described for use inretroviral targeting (7-9). In this embodiment, the retroviral packagingcell line may also display the growth factor-envelope protein fusion onthe surface of the retroviral packaging cell line. The chimeric envelopecould be expressed as the sole viral envelope protein in an attempt totarget the retrovirus to stem cells, as well as to transduce a growthsignal, or in concert with the “wild type” envelope protein, to inducegrowth in growth factor responsive target cells, without targeting to aspecific cell type. The former strategy is more applicable to an in vivosituation, the latter to an in vitro transduction process. An example ofthis is the expression of the growth factor as a fusion with viralenvelope SU protein of murine leukemia virus (MLV).

In some instances, expressing the growth factor as a fusion with a viralenvelope protein, may lead to the nucleic acid encoding the polypeptidenot being incorporated into the genome of the target quiescent cells.This can be overcome by introducing a cleavable linker between the viralenvelope protein and the growth factor so that the growth factor can becleaved from the viral particle by addition of a cleaving agent,typically once the quiescent cells start dividing. An example of such asystem is the use of a chimeric envelope protein in which viral envelopeprotein is linked to a factor X_(a) linker which is in turn linked tothe growth factor. In this system, factor X_(a) protease can be used tocleave the growth factor from the surface of the viral particles, sothat the particles can transfer the nucleic acid encoding thepolypeptide to the target cells where it can be incorporated into theirgenomes.

Preferably, the surface bound growth factor is FLT3-ligand, or stem cellfactor, also known as mast cell growth factor, kit ligand factor orSteel factor. Nucleic acid sequences encoding stem cell factors aredescribed in WO92/00376, e.g. the Δ28 MGF stem cell factor.

Preferably, the vector is a retroviral vector such as MFG or the pBabevector series. Alternatively, present invention could employ alentiviral vector producer cell line. In the viral display aspect of theinvention, as it is known that the envelope glycoproteins of lentiviralvectors can be substituted by the envelope proteins of C-typeretroviruses, the chimeric envelope glycoproteins described below couldbe used with lentiviral vectors such as those based on HIV, CAEV orVisna. Further vectors suitable for use in the methods described hereincan be readily identified by the skilled person.

Typically, the desired protein or polypeptide will be one that a patientis unable to synthesise in his or her body or does not synthesise in theusual amount. An example of this is the use of gene therapy to treatadenosine deaminase dependent severe combined immunodeficiency(ADA-SCID). However, the concepts described herein are applicable tosituations in which the nucleic acid encodes a protein or polypeptidethat binds a substance that is overexpressed in a patient's body, e.g.causing some harmful physiological effect, or a protein or polypeptidethat can bind to a polypeptide that is produced in a patient's body inan inactive form to activate it or in an active form to inactivate it.Preferably, the use of the present invention in these applications hasthe advantage that the therapy provided by transfecting the stem cellsis long lasting or permanent, thereby helping to avoid the need forfrequently repeated treatment.

Diseases that might be treated using the methods and materials describedherein include all forms of chronic granulomatous disease (CGD), allforms of severe combined immunodeficiency (SCID), hyper gammaglobulinaemia syndrome (hyper IgM), Wiskott-Aldrich Disease (WAS),thallassaemia, sickle-cell anaemia, other anaemias due to deficienciesof red blood cell proteins, neutrophil defects due to failure tosynthesise granule components, e.g. myeloperoxidase deficiency,haemophilia and other clotting disorders such as complementdeficiencies, lysomal storage disorders, such as Gaucher's disease,Hurler's disease, and mucopolysaccharidosis, leukocyte adhesiondeficiency (LAD), bare lymphocyte syndrome, cancer and AIDS.

Other applications of the invention include the genetic modification ofhaematopoietic stem cells to repopulate the immune system withgenetically modified T-lymphocytes that resist HIV, the geneticmodification of haematopoeitic stem cells to repopulate bone marrow withhaematopoietic progenitors that resist the myelosuppressive effects ofcytotoxic chemotherapy, and the genetic modification of T-lymphocyteswith chimeric T-cell receptors to target cytotoxic T-cells againsttumours or virally infected cells.

In a further aspect, the present invention provides compositionscomprising a retroviral packaging cell line or retroviral particles setout above, in combination with a suitable carrier. In this aspect, thepresent invention provides pharmaceutical compositions suitable fordelivering nucleic acid encoding a desired polypeptide to a populationof stem cells in vitro, e.g. to prepare engineered stem cells forsubsequent implant into a patient. Alternatively, the composition couldbe used in vivo, to directly deliver the nucleic acid to a patient's ownstem cells. In this case, the composition preferably comprises aretroviral vector incorporating the nucleic acid encoding a desiredprotein or polypeptide and displaying a growth factor on its surface,e.g. as part of an envelope protein.

In a further aspect, the present invention provides the use of aretroviral cell line or retroviral particles as described above in thepreparation of a medicament for treating a disease or disorder thatresponds to the polypeptide encoded by the nucleic acid packaged in theretroviral particles.

In this aspect, preferably the medicament comprising the retroviralpackaging cell line or retroviral particles is administered byimplantation into a patient's bone marrow or is administered by infusioninto a patient's blood. In order to allow the packaging cells to targetthe bone marrow when administered by infusion, advantageously, receptorssuch as integrins can be expressed on the surface of the cells.Alternatively or additionally, the immunogenicity of the packaging cellscan be reduced by expressing an immunosuppressive factor such asFAS-ligand on the cell surface which can bind to activated T-cellFAS-receptors, triggering the T-cells to die by apoptosis. FAS-ligandexpressing allogeneic cell implants have previously been shown to resistimmune mediated rejection.

In a further aspect, the present invention provides a method oftransforming a population of quiescent cells with nucleic acid encodinga polypeptide so that the nucleic acid is incorporated into the genomeof the cells, the method comprising exposing the cells to a retroviralpackaging cell line or retroviral particles as described above, whereinthe surface bound growth factor induces the cells to divide, so that thenucleic acid encoding the polypeptide for treating a disease or disordercontained in the viral particles can incorporate into the genome of thecells.

In this aspect, preferably the quiescent cells are a population of bonemarrow cells enriched in haematopoeitic stem cells.

In further aspects, the present invention provides a population of cellsproduced by the above method having the nucleic acid encoding apolypeptide for treating a disease or disorder stably incorporated intotheir genome, and pharmaceutical compositions comprising thesepopulations of cells.

In a further aspect, the present invention provides a method forintroducing nucleic acid encoding a polypeptide for treating a diseaseor disorder into the genome of a population of cells in viva, the methodcomprising administering a retroviral packaging cell line or retroviralparticles by implantation into a patient's bone marrow or by infusioninto a patient's blood.

By way of example, the present invention will now be described in moredetail with reference to the accompanying figures. The followingexamples are provided to illustrate the present invention, and shouldnot be interpreted as limiting the scope of the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A) top panels, bone marrow cells following 5 days incubationwith 5-FU (right) or without 5-FU (left); bottom panels, staining ofcells as above for SCF receptor at completion of 7 days selection in5-FU (right) or without 5-FU (left). B) PCR analysis of colonies arisingfrom retrovirally transduced, 5-FU selected, stem cells in semi-solidmedium following 4 weeks long term culture. 1-9, colonies; N, negativecontrol; C, positive control; M, size markers. The arrow indicates theretroviral PCR product.

FIG. 2. Tritiated thymidine labelling of 5-FU selected cells. Bonemarrow cells were incubated as described earlier for 7 days in 5-FU [A],or not [B], after which tritiated (³H) Thymidine was added to the mediumand the cells incubated for a further 16 hrs. Following this incubationthey were pelleted onto glass microscope slides using Cytospin (ShandonInstruments). The slides were dipped in photographic emulsion (Ilford)and allowed to dry before incubation in the dark at −70° C. for oneweek. The slides were then developed using standard developer and fixerand counter stained with Wright's stain. Cells undergoing division arelabelled by the incorporation of ³H thymidine into DNA, which leads tothe formation of silver grains in the emulsion. The 5-FU treated cells(panel A) show no labelling indicating quiescence, whereas the untreatedcells (panel B) show extensive and intense labelling indicative ofactive cell division.

FIG. 3 shows the restriction map of plasmid pJP2 carrying SCF cDNA.

FIG. 4 shows schematically the construction of retroviral packaging celllines expressing membrane-bound SCF.

FIG. 5 shows the proliferative response of TF-1 cells to soluble SCF andthe membrane bound growth factor. To start this analysis we titratedsoluble SCF to against TF-1 cells to establish the optimum concentrationof SCF

FIG. 6 shows the results of a proliferation assay on TF-1 cellsdemonstrating that TF-1 cells have a proliferative response to the AM12cell line alone due to the production of IL6 by the cell line.

FIG. 7 shows the titration of anti-IL6 antibody and its effect on theproliferative response of TF-1 cells incubated with AM12 cells only,demonstrating that it is possible to test for SCF-mediated transductionof quiescent TF-1 cells co-cultured with retroviral producer cells byblocking the IL6-dependent proliferation using anti-IL6 antibody.

FIG. 8 shows a diagrammatic representation of the plasmid constructs(SCFA1, SCFAX1, FLA1, and FLAX1, SEQ ID NOs:1-4, respectively) used inexample 4 in the production of retrovirus displaying surface boundgrowth factor as an N-terminal extension of the viral envelope SUprotein.

FIG. 9 is a Western blot showing the chimeric envelope proteins of SCFand FL displaying viruses.

FIG. 10 is a Western blot showing specific cleavage at the factor X_(a)cleavage site of SCFAX1 that upon treatment of the viral pellets withFX_(a) protease.

FIG. 11 shows the effect of SCF competition on tropism of retroviralvectors displaying SCF.

FIG. 12 shows tropism of retroviral vectors displaying cleavable andnon-cleavable SCF for Kit positive cells.

FIG. 13 shows a schematic representation of the transduction of stemcells using retroviral packaging cell lines expressing membrane boundSCF.

DETAILED DESCRIPTION

Materials and Methods

Cell Culture

Bone marrow cells were harvested and stem cells selected using the 5FUtechnique transduced with retroviral vectors and incubated in long termbone marrow culture all as described in the original application. TF-1cells were maintained in RPMI medium supplemented with 10% fetal calfserum, 2 mM glutamine, penicillin and streptomycin. Recombinant humanGM-CSF was added to 200 pg/ml for routine passage and recombinant humanSCF at 25 ng/ml was used for short term growth support. Packaging andproducer cell lines were cultured under the conditions described belowand irradiated (500 rads) prior to use in culture experiments.

PCNA Staining

Staining of cells for proliferating cell nuclear antigen (PCNA) wasperformed on cells spun onto microscope slides using a Cytospin (ShandonInstruments) and fixed in methanol, using a mouse anti-human monoclonalantibody (Dako) directly conjugated to FITC. Cells were incubated for 1hour at room temperature before the antibody solution was washed awaywith PBS, the slides air-dried, cover-slipped using Citifluor and thefluorescence viewed under UV light using a Zeiss microscope.

Construction of LacZ Retroviral Producer Cells Expressing Surface SCF

A retroviral producer line established in the packaging line AM12 andcontaining the retroviral vector genome nlsLacZ (12) was used as atarget to introduce the plasmid pJP2 by calcium phosphate transfectionas described in the original submission. Cells were selected for thepresence of the SCF-encoding plasmid using the linked histinidolresistance marker, as described previously.

Proliferation Assays

Cells were washed twice in PBS pelleted and resuspended in media withoutgrowth factor and incubated overnight. Cytokines and ³H thymidine (10μCi) were added to the media and the cells incubated for a further 24hours. The cells were then harvested using a Titertek cell harvester andthe incorporated radioactivity counted using a scintillation counter.

X-gal Staining

For assessing retroviral gene transfer, TF-1 cells were deposited ontomicroscope slides using a Cytospin (Shandon Instruments) and fixed inPBS buffer containing 0.5% glutaraldehyde. β-galactosidase activity wasdetected by staining the cells in situ with 0.1% X-gal(5chloro-4bromo-3indolyl-β-D-galactopyranoside.) in PBS buffercontaining, 0.01% sodium deoxycholate, 0.02% NP40, 2 mM MgCl₂, 5 mMpotassium ferricyanide and 5 mM potassium ferrocyanide for 1-2 hours at37° C.

Tritrated Thymidine Labelling

For ³H thymidine labelling TF-1 cells were transduced with retroviralvector as described above in RPMI media to which 10 μCi of ³H thymidinewas added. After overnight labelling, the cells were deposited ontomicroscope slides and stained for β-galactosidase activity before beingwashed dried and dipped in photographic emulsion. The emulsion wasallowed to air dry overnight and the slides were exposed at −70° C. in alight tight box for 3 days. To visualise the ³H thymidine incorporationthe emulsion was developed and fixed using standard X-ray film developerand fixer and cells producing silver grains were assessed by light anddark field light microscopy.

EXAMPLE 1

Retroviral Transduction of Quiescent Target Cells with RetroviralProducer Cells Expressing Surface Growth Factor (SCF); the Establishmentof a Model System

To demonstrate the feasibility of using producer cells that expresssurface growth factor to transduce quiescent cells with retroviralvectors, we have developed a model system using the growthfactor-dependent cell line TF-1 (12) . This cell line was developed froman erythroleukaemic patient and probably equates to cell arrested at anearly stage of megakaryocyte development. The cells can only be grown inthe presence of growth factors, the most usual being IL-3 or GM-CSF towhich they respond very sensitively. They also are capable of dividingin response to SCF though this response is significantly weaker. Thesecells can easily be rendered quiescent by withdrawal of growth factorsupport. We used these cells as targets for retroviral transduction,following induction of quiescence.

In order to facilitate this analysis, we constructed a retroviralproducer line expressing surface SCF (see FIG. 4). This was done bytransfecting the same plasmid we had constructed before containing thehSCF cDNA (plasmid pJP2, FIG. 3), into retroviral producer cellstransducing a retroviral vector encoding the gene for β-galactosidase(NLSlacZ). This bacterial enzyme can be used in conjunction withsynthetic substrates to produce a blue staining reaction when activeenzyme is present. Accordingly, in these experiments, successfullytransduced cells will stain blue following retrovirally mediated genetransfer. The resulting producer line was identified as LacJP.Immunofluorescent staining of the LacJP cell line with anti-SCF antibodyshowed the presence of the surface bound SCF.

We compared the proliferative response of TF-1 cells to soluble SCF andthe membrane bound growth factor. To start this analysis we titratedsoluble SCF to against TF-1 cells to establish the optimum concentrationof SCF (FIG. 5). TF-1 cells were then grown in the presence of solublerecombinant human SCF or in the presence of the lac JP cell line or theparent packaging cell line (AM12). These initial experiments quicklyrevealed, however, that the retroviral packaging cell line produced agrowth factor that was a potent mitogen for the TF-1 target cells (FIG.6) . It was previously described in the literature that packaging cellslines were sources of the cytokine IL-6. We hypothesised that mitogensecreted by the retroviral packaging cells might be IL-6. To test thiswe attempted to block the response to IL-6 in our cultures by theaddition of neutralising antibody to the cytokine. Titration of theneutralising antibody (FIG. 7) demonstrated that it was possible tocompletely eradicate the IL-6 mediated proliferative response of TF-1cells when co-cultured with retroviral producer cells. All thesubsequent experiments were therefore performed in the presence ofinhibitory amounts of anti-IL-6.

We then tested the ability of our modified producer cells to facilitatetransduction of quiescent target cells. TF-1 cells in exponential growthwere removed from growth factor and incubated overnight to allow them tobecome quiescent. TF-1 cells were then co-cultured overnight with theretroviral producer cells expressing surface SCF or the parent producerline. Following co-culture, the TF-1 cells were removed collected ontomicroscope slides and stained for β-galactosidase production. Cells thatwere co-cultured on the parent producer line showed no evidence forretroviral transduction. In contrast, approximately 3% of cells thatwere co-cultured with the SCF producer cells were found to be positivelystaining. These were confirmed as cycling TF-1 cells by doubly-labellingthe cells with titrated thymidine (³H). This radioactive nucleosidebecomes incorporated into the DNA of dividing cells and can be detectedby autoradiographic deposition of silver grains in a photographicemulsion into which the slides have been dipped. As expected, many ofthe transduced TF-1 cells also showed the presence of silver grainsindicating that cell division had taken place.

Thus, these experiments show that expression of a surface bound growthfactor by retroviral producer cell lines is able to facilitate theretroviral transduction of a quiescent target cell population andtherefore enable the retroviral-mediated transfer of genes to cells thatwould normally be refractory to this technique.

EXAMPLE 2

Retroviral Transduction using Populations of Cells Isolated fromUmbilical Cord Blood

We carried out a transduction experiment similar to those describedabove using haematopoietic progenitor cells obtained from humanumbilical cord blood. Progenitors were selected from cord blood usingthe Macs™ (Miltenyi Biotech) system to isolate CD34+ progenitor cells(15). The CD34+ population in cord blood is made up extensively ofquiescent cells (13, 16). These cells were transduced in vitro asdescribed above by co-culture for 48 hours in the presence of thenlsLacz producers or the LacJP producers. The cord blood cells were thenharvested and pelleted onto microscope slides and stained forβ-galactosidase activity as described above. Cells that had been exposedto the nlsLacZ producers had a low proportion of blue-staining cells(<10%), whereas those that had been exposed to the LacJP producers thatexpresses SCF on its surface had a very high proportion of blue-stainedcells (>80%) . In addition, blue-staining colonies formed by subsequentgrowth of these populations of cells in semisolid media were only foundin cultures derived from cells that had been co-cultured with the LacJPcell line.

EXAMPLE 3

Retroviral Transduction of Haematopoietic Stem Cells using a RetroviralPackaging Cell Line Expressing Surface Bound SCF

(a) Production of the Retroviral Packaging Cell Line

The cell line 1MI-ΔSCF was constructed as follows: the parent producercell line 1MI was derived from the Am12 packaging cell line (1), bycalcium phosphate-mediated DNA transfection, using the retroviral vectorencoding the p47-phox cDNA we described previously (2), with theexception that the neomycin resistance cassette was removed. Theretroviral backbone is derived from the pBabe series of vectorsdescribed by Morgenstern et al (3). High titre producer clones were thenselected by “dot blot” analysis of successful transfectants. The 1MIproducer line was then transfected as described above using the plasmidpJP2 (FIG. 3) encoding the membrane-associated form of the human stemcell factor (SCF). Cells expressing SCF were selected using histidinol.Individual clones were grown out and tested for expression of SCF byimmunofluorescence with a labelled anti-SCF antibody. The plasmid pJP2was constructed by insertion of an 816 bp HindIII to BamHl, SCF cDNAfragment into the mammalian cell expression plasmid pREP8 (InvitrogenCorp). The SCF cDNA was excised from the plasmid BSSK: huMGFΔ28, seeWO92/00376.

(b) Selection and Transduction of PHSC

Bone marrow cells (10 mls; approx 5×10 ⁷ cells) were aspirated from theiliac crest of normal volunteers under local anaesthesia. The cells werewashed twice with sterile PBS, re-pelleted and layered onto the surfaceof a discontinuous ficoll gradient. Cells were separated bycentrifugation for 20 mins at 2500 rpm. Mononuclear cells were removedfrom the interphase and washed with PBS. Cells were then incubated inIscove's DMEM medium supplemented with 10% fetal calf serum,5-fluorouracil (5-FU), stem cell factor (SCF) and IL-3, as described byBeradi et al (Science 267 1995) Following seven days in selection (seeFIG. 1A) , the surviving cells were co-cultured for 48 hrs in thepresence of the SCF-producer line. Following co-cultivation, they wereremoved from the producers and used to establish long term cultures(LTC) on heterologous irradiated human stroma, in McCoy's mediummodified for long term culture. After 4 weeks in LTC, cells were platedin semi-solid media containing cytokines (StemGEM™), to allow coloniesto develop.

(c) Detection of Transduced Cells

Transduction was scored by PCR analysis of colonies (FIG. 1B). The PCRrelied on a nested strategy using two upstream and one downstreamprimers. An initial round of 35 cycles of amplification using the mostupstream primer and the downstream primers was performed. A smallaliquot of this reaction was removed and re-amplified in a secondreaction using the second upstream primer and the downstream primer. Themost upstream primer is complementary in sequence to a region from thegag gene of the retroviral vector and the other two primers arecomplementary to different regions from the p47-phox cDNA sequence. Thesize of the initial product is 454 nucleotides and the nested product180 nucleotides. This strategy ensures that the PCR product is specificfor the retrovirally encoded p47-phox gene and not the endogenous gene.The products of the PCR amplification were visualised under ultra-violetlight (300 nm) following separation by standard agarose gelelectrophoresis on 2% gels containing ethidium bromide.

TABLE 1 PCR Colony Data (Ethidium Bromide Staining) Fold increaseExperi- +Colonies/Total +Colonies/ % + % + with ment SCF Total ControlSCF Control SCF 1 5/20 — 25 — — 2 5/30 0/30 17 0 >5  3* 11/30 1/30 37 311 4 19/30 7/20 63 35  2 5 5/30 — 17 — — 6 4/30 0/30 13 0 >4 *Expt 2re-evaluated using Southern Blot hybridisation.

The data set out in table 1 shows that in five separate experiments onmarrow cell cultures indicated that is approximately 13% and 63% ofcolonies were positive for the presence of the retroviral genome,showing that the retroviral vector had succeeded in delivering thep47-phox gene to the PHSCs.

In addition, the data presented as experiment 2 was re-evaluated using adifferent, more sensitive, method (Southern blotting), demonstrating an11-fold increase in the rate of transduction of the 5FU-selected PHSCfollowing exposure to the retroviral producers that express stem cellfactor on their surface. As no positive colonies were obtained from thecontrols in two experiments (Experiments 2 and 6), the increased ratesof transduction can only be expressed as greater than 5 or 4 timesbetter than the control respectively. These values can therefore beconsidered as the absolute minimum in terms of the benefit of using theSCF-expressing producers; the actual value may be considerably greater.

(iv) PCNA Staining

A nuclear antigen referred to as “proliferating cell nuclear antigen”(PCNA) a component of the DNA replication machinery can be used as amarker of cells in the process or capable of undergoing cell division.As such, staining cells with antibody to this antigen can giveinformation on the cycling status of cells in a chosen population.Staining of PHSC selected by the 5FU technique with a monoclonalantibody to PCNA (PC10), revealed that the majority of the cells (˜90%,FIG. 1a and Table 2) were unlabelled and were therefore quiescent, asexpected. These cells were then cultured for 48 hours in the presence ofthe SCF-expressing producer line or the control parent cell line andstained with the PC10 antibody. Cells that had been exposed to the SCFproducers showed a marked increase in the fraction of positivelystaining cells (˜50% and table 2).

TABLE 2 PCNA staining Total + % + Cells +cells* cells cells 5FU cells8/73 3/42 11/115 9.5 Post SCF 9/17 9/18 18/35 51.4 Post Control 0/10 —0/10 0 *Cells were counted from two different random views undermicroscopical examination. Insufficient control cells were obtained toenable two counts to be made.

EXAMPLE 4

Production of a Retrovirus Displaying Surface Bound Growth Factor as anN-terminal Extension of the Viral Envelope SU Protein

(a) Construction of Chimeric Envelope Expression Plasmids

Plasmids were created encoding chimeric envelopes in which stem cellfactor (SCF) or Flt3 ligand (FL) is fused to the first codon of the SUenvelope glycoprotein as a factor Xa cleavable or non-cleavableN-terminal extension of the 4070A (amphotropic) murine leukaemia virus(MLV).

SCF and FL cDNA was PCR amplified and tailed with Sfil and Not 1restriction sites. The PCR products were cloned into existing chimericenvelope expression plasmids EA1 and EXA1 (10) after digestion with therestriction enzymes Sfil and Not 1. FIG. 8 shows a diagrammaticrepresentation of the plasmid constructs. The sequences of all theconstructs was confirmed by dideoxysequencing.

(b) Production of Viruses

The chimeric envelopes and control amphotropic (4070A) envelopes wereexpressed in TelCeB.6 complementing cells, which express MLV gag-polcore particles and a nlsLacZ retroviral vector. Envelope expressionplasmids were transfected by calcium phosphate precipitation into theTelCeB.6 cells. Transfected cells were selected with phleomycin andblasticidin in DMEM supplemented with 10% foetal calf serum (FCS) andgrown to confluency. Viral supernatants were harvested from stablytransfected cells after overnight incubation in either serum free DMEMor DMEM containing 10% FCS and filtered with a 0.45 μm filter for use ininfection or binding experiments. For immunoblotting, viral supernatantswere filtered (0.45 μm) and then pelleted by ultracentrifugation at30,000 rpm in a SW40 rotor for one hour at 4° C. The pelleted viralparticles were re-suspended in 100 μl of phosphate buffered saline andstored at −20° C.

(c) Target Cells

The murine cell line NIH 3T3 was grown in DMEM supplemented with 10%FCS. The human SCF receptor (Kit) expressing cell line HMC-1 was grownin Iscove's modified Dulbecco's eagle medium (IMDM) supplemented with10% FCS and monothioglycerol. The human kit negative cell line K422 wasgrown in RPMI supplemented with 10 FCS.

(d) Immunoblots

Ten μl of the pelleted viral particles were separated on a 10%polyacrylamide gel under reducing conditions and subsequentlytransferred to nitrocellulose. The viral SU proteins were detected usinga primary goat anti-envelope antibody. Blots were developed using asecondary anti-goat antibody conjugated to horseradish peroxidase and anenhanced chemiluminescence kit.

(e) Infections

Target cells were plated into six-well plates at approximately 10⁵ cellsper well and incubated overnight at 37° C. (adherent cells) or platedinto six well plates at approximately 10⁶ cells per well one hour beforeinfection (suspension cells). Filtered viral supernatant in serum freemedium was added to the target cells and incubated for 4 hours in thepresence of 8 μg/ml polybrene.

The retroviral supernatant was then removed from the target cells, themedium was replaced with the usual medium and the cells were placed at37° C. for a further 48-72 hours. X-gal staining for detection ofβ-galactosidase activity was carried out as previously described (11).Viral titre was calculated by counting blue stained coloniesmicroscopically and expressed as enzyme forming units per ml (adherentcells) or percentage blue stained cells (suspension cells).

(f) Immunoblotting of Pelleted Virus

The viral pellets of both SCF and FL displaying viruses were analysed byimmunoblotting and a representative blot is shown in FIG. 9. Thepresence of chimeric viral envelope with a distinct mobility from thatof the wild type virus is demonstrated. FIG. 10 shows that upontreatment of the viral pellets with 4 μg/ml FX_(a) protease, the FX_(a)cleavage signal in the interdomain linker of the expressed envelopeSCFXA1 is correctly recognised and cleaved to yield a SU cleavageproduct with identical mobility to the unmodified 4070A envelope SU.

(g) Host Range Properties of Vectors Incorporating the ChimericEnvelopes

Viral supernatant harvested from cells transfected with the variousconstructs was used to infect mouse fibroblasts. Gene delivery to the3T3 cells was demonstrated for the amphotropic chimeras and wasunaffected by protease cleavage. Thus viral infectivity mediated by theunderlying envelope was not blocked by display of these dimeric ligands.

The infectivity of viruses incorporating the SCFA1 and SCFXA1 chimericenvelopes was tested on HMC-1 cells—a human Kit expressing cell line.Both vectors were capable of binding to Kit positive cells but gave verylow titres compared to the wild type vector. When soluble SCF was addedas competitor to prevent the vectors from binding to Kit, theinfectivity was increased, suggesting that the reduced ability of thesevectors to bind to Kit positive cells was a consequence of targetedbinding to SCF receptors (FIG. 11). These vectors were then treated withfactor X_(a) protease prior to infection. Pre-incubation with FXa had noeffect on the titre of the SCFA1 vector but restored the titre of SCFXA1to that of the wild-type amphotropic vector (FIG. 12). This effect ofFX_(a) cleavage did not occur on the Kit negative cell line K422. Thisdata provides evidence that retroviral vectors displaying SCF arecapable of selectively binding to human cells expressing Kit but thatinfection of Kit positive cells cannot take place until the engineeredbinding domain has been cleaved. In this respect, this ligand-receptorsystems resembles the EGF system developed in our laboratory (11) and ispotentially amenable to the two-step targeting strategies developed forEGF.

Discussion

The above results show that good levels of transduction of PHSC can beachieved using engineered retroviral packaging cells expressing humanSCF on their cell surface. Thus, the results above indicate that thecells should be capable of simultaneously delivering both a growthsignal and a retroviral vector to the target PHSC. This simultaneousdelivery of vector and growth signal should also have the advantage ofincreasing the effective retroviral titre, owing to the intimateassociation of producer and target cells.

SCF has been shown to have both soluble and membrane-bound forms.Evidence acquired from the study of mice carrying a small intragenicdeletion in the gene encoding the SCF receptor has indicated that themembrane-bound form of the cytokine is essential for normalhaematopoiesis. Despite being able to synthesise a soluble SCF retainingfull biological activity, these mice are as badly affected as theircounterparts who carry a complete deletion of the gene. While notwishing to be bound by any particular theory, we believe that the invivo biological activities of the soluble and membrane-associated formsof the growth factor are distinct, and that normal haematopoiesis has anabsolute requirement for the membrane-bound form of SCF that cannot besubstituted by the soluble form. It may also be that the expression ofbound SCF on the cell surface changes/reduces the extent to which othergrowth factors are expressed, and that this has a beneficial effect ontransduction levels of the PHSC.

Improved transduction rates using the method may be achieved using thesynergistic action of additional cytokines. In this regard, SCF isparticularly noted for its property of interacting in this way withother growth factors, which has led to the suggestion that on its own itmay not be a mitogen but acts as an anti-apoptotic factor. To assessthis, similar experiments to those described above can be performedusing additional cytokines added to the media in conjunction with ourmodified producers. Ideally, we would hope to find conditions favouringself-renewal at the expense of differentiation. This would have thehighly desirable consequence of enabling us to expand PHSC numbers inculture. One factor thought possibly to act in this way is MIP1-α1.There is also evidence that stem cell quiescence may be negativelyinfluenced by TGF-β, antagonists of this molecule may therefore bebeneficial in stimulating cells into cycle. Of the positively actingcytokines, LIF, the factor that blocks differentiation of mouseembryonal stem cells and IL-11, a recently identified member of the samefamily of cytokines, are candidates for acting on stem cells, as is flt3ligand, a molecule with a similar spectrum of activities to SCF.

As regards embodiments of the invention using growth factors displayedon the surface of retroviruses, the results presented above demonstratethat the dimeric ligands SCF and FL can successfully be displayed onretroviruses and that these ligands retain the capability of binding totheir receptors. We have demonstrated that, although infection cannot bemediated through the displayed ligand, if a protease cleavable linker isused to fuse the displayed ligand to the viral envelope, targetedinfection can result. The modified viruses described here retain theircapacity to infect cells through the Ram1 receptor but other resultsfrom our laboratory indicate that it is possible to construct acleavable linker which is capable of blocking infection through thenatural viral receptor. In should therefore be possible to targetretroviral gene transfer specifically to PHSC.

The above method describes a protocol which is potentially applicable toany clinical procedure requiring the transfer of genetic information topluripotent haematopoeitic stem cells (PHSC). As discussed above, thismethod is applicable for gene therapy of inherited haematopoeiticdisorders, such as the immunodeficiencies, but it could also beapplicable to conditions such as haemophilia, or other conditionsrequiring the synthesis of a pharmacologically active compound normallypresent in the serum. There are also potential applications in the fieldof cancer therapy, primarily as a way of protecting cells from cytotoxicagents or radioprotecting them, thus giving them a survival advantageover non-treated bone marrow cells.

References

The references mentioned in this application are all herein incorporatedby reference.

1. Markowitz, D., S. Goff, and A. Bank. 1988. Construction and use of asafe and efficient amphotropic packaging cell line. Virology 167:400.

2. Thrasher, A., M. Chetty, C. Casimir, and A. W. Segal. 1992.Restoration of Superoxide Generation to a Chronic GranulomatousDisease-Derived B-Cell Line by Retrovirus Mediated Gene Transfer. Blood80:1125.

3. Morgenstern, J. P. and H. Land. 1990. Advanced mammalian genetransfer: high titre retroviral vectors with multiple drug selectionmarkers and a complementary helper-free packaging cell line. NucleicAcids Res. 18:3587.

4. Anderson, D. M., D. E. Williams, R. Tushinski, S. Gimpel, J.Eisenman, L. A. Cannizzaro, M. Aaronson, C. M. Croce, K. Huebner, and D.Cosman. 1991. Alternate splicing of mRNAs encoding human mast cellgrowth factor and localisation of the gene to chromosome 12q22-12q24.Cell Growth Differ 2:373.

5. Beradi, A. C., A. Wang, J. D. Levine, P. Lopez, and D. T. Scadden.1995. Functional isolation and characterization of human hematopoieticstem cells. Science 267:104.

6. Mullis, K., F. Faloona, S. Scharf, R. Saiki, G. Horn, and H. Erlich.1986. Specific enzymatic amplification of DNA in vitro: the polymerasechain reaction. Cold Spring Harb Symp Quant Biol 51:263.

7. Russell, S. J., R. E. Hawkins, and G. Winter, 1993. Retroviralvectors displaying functional antibody fragments.

8. Valesia-Wittman, S., A. Drynda, G. Delange, M. Aumailley, J. M.Heard, O. Danos, G. Verdier, and F. L. Cosset. 1994. Modifications inthe binding domain of avian retrovirus envelope protein to redirect thehost range of retroviral vectors. J. Virol., 68:4609.

9. Kasahara, N., A. M. Dozy and Y. N. Kan. 1994. Tissue-specifictargeting of retroviral vectors through ligand-receptor interactions.Science, 266:1373.

10. Nilson B H K, Morling F J, Cosset F-L, Russell S. J. Targeting ofretroviral vectors through protease-substrate interactions. Gene Therapy1996, 3:280-286.

11. Takeuchi Y et al. Type C retrovirus inactivation by human complementis determined by both the viral genome and the producer cell. J Virol1994, 68: 8001-8007.

12. Bagnis, C., Gravis, G., Imbert, A. M., Herrera, D., Allario, T.,Galindo, R., Lopez, M., Pavon, C., Sempere, C., and Mannoni, P. (1994).Retroviral transfer of the nlsLacZ gene into human CD34+ cellpopulations and into TF-1 cells: future prospects in gene therapy. Hum.Gene Ther., 5, 1325-1333.

13. Broxmeyer, H. E., Lu, L., Cooper, S., Ruggieri, L., Li, Z. H., andLyman, S. D., (1995). Flt3 ligand stimulates/costimulates the growth ofmyeloid stem/progenitor cells. Exp. Hematol., 23, 1121-1129.

14. Einerhand, M. P., Bakx, T. K., and Valerio, D., (1991). IL-6production by retrovirus packaging cells and cultured bone marrow cells.Hum. Gene Ther., 2, 301-306.

15. Krause, D. S., Fackler, M. J., Civin, C. I., and May, W. S., (1996).CD34: structure biology and clinical utility, Blood, 87, 1-13.

16. Leitner, A., Strobl, H., Fischmeister, G., Kurz, M., Romanakis, K.,Haas, O. A., Printz, D., Buchinger, P., Bauer, S., Gadner, H., andFritsch, G. (1996). Lack of DNA synthesis among CD34+ cells in cordblood and in cytokine mobilized blood. Br. Haematol 192, 255-262.

4 1 11 PRT Artificial Sequence plasmid construct 1 Ser Ser Leu His AlaAla Ala Met Ala Glu Ser 1 5 10 2 15 PRT Artificial Sequence plasmidconstruct 2 Ser Ser Leu His Ala Ala Ala Ile Glu Gly Arg Met Ala Glu Ser1 5 10 15 3 11 PRT Artificial Sequence plasmid construct 3 Pro Thr AlaPro Ala Ala Ala Met Ala Glu Ser 1 5 10 4 15 PRT Artificial Sequenceplasmid construct 4 Pro Thr Ala Pro Ala Ala Ala Ile Glu Gly Arg Met AlaGlu Ser 1 5 10 15

What is claimed is:
 1. A method of transforming a quiescent cell with anucleic acid encoding a polypeptide, the method comprising: exposingsaid quiescent cell in vitro to a retroviral packaging cell, saidretroviral packaging cell comprising a retroviral vector and anexogenous nucleic acid encoding a growth factor, wherein said growthfactor is displayed on the surface of said retroviral packaging cell,wherein said retroviral vector comprises said nucleic acid encoding saidpolypeptide, and wherein said growth factor displayed on the surface ofsaid retroviral packaging cell induces said quiescent cell to divide, sothat the nucleic acid encoding said polypeptide can incorporate into thegenome of said quiescent cell.
 2. The method of claim 1 wherein saidquiescent cell is a hematopoietic stem cell.
 3. The method of claim 1wherein the growth factor is stem cell factor (SCF) or FLT3 ligand. 4.The method of claim 1 wherein said retroviral packaging cell displayingsaid growth factor on its surface displays multiple growth factors. 5.The method of claim 1 wherein said growth factor is expressed as anN-terminal fusion protein with a retroviral envelope protein.
 6. Themethod of claim 1 wherein said growth factor is expressed as a fusionprotein with a viral envelope protein and is fused to the envelopeprotein via a cleavable linker.
 7. The method of claim 5 wherein saidretroviral envelope protein is viral envelope SU protein.
 8. The methodof claim 1 wherein said retroviral packaging cell displaying said growthfactor on its surface further expresses nucleic acid encoding a receptorto target the retroviral packaging cell to the bone marrow and/or animmunosuppressive factor so that the receptor and/or immunosuppressivefactor are displayed on the cell surface of said retroviral packagingcell.